Lactobacillus transformation (Speer 2012)

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(Procedure)
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*0.5M Sucrose, 10% glycerol solution
*0.5M Sucrose, 10% glycerol solution
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==Procedure==
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°C==Procedure==
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#Grow L. plantarum overnight from freezer stock in 5ml MRS medium (30°C without shaking).
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#'''Grow ''L. plantarum'''''
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#Add 1g glycine to two flasks of 50ml MRS medium to make 100ml 2% glycine.
+
##Inoculate 5ml MRS medium with ''L. plantarum'' freezer stock.
 +
##Grow overnight at 30°C without shaking.
 +
#'''Make 100ml 2% glycine:'''
 +
##Add 1g glycine to two flasks of 50ml MRS medium.
##Shake to dissolve.
##Shake to dissolve.
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#Add 1ml overnight culture to each flask (1:50 dlution)
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#'''Add 1ml overnight culture to each flask (1:50 dlution).'''
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#Culture for ~3 hours.
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#'''Culture for ~3 hours at 37°C with shaking.'''
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#'''Wash 2x with ice-cold DI water:'''
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##Centrifuge culture for 5min at 4000g
 +
##Resuspend in 25ml ice-cold DI water.
 +
##Repeat.
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#'''Treat with ice-cold EDTA:'''
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##Centrifuge for 5min at 4000g
 +
##Resuspend in 10ml 50mM EDTA.
 +
##Incubate on ice for 5 minutes
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#'''Wash with ice-cold DI water:'''
 +
##Centrifuge culture for 5min at 4000g
 +
##Resuspend in 25ml ice-cold DI water.
 +
#'''Wash 2x with Electroporation Buffer:'''
 +
##Centrifuge culture for 5min at 4000g
 +
##Resuspend in 25ml ice-cold 0.5M Sucrose, 10% glycerol.
 +
##Repeat.
 +
#'''Concentrate cells:'''
 +
##Centrifuge culture for 5min at 4000g
 +
##Resuspend in 0.8ml ice-cold 0.5M Sucrose, 10% glycerol.
 +
#'''Divide cells:'''
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##Aliquot 90μL of cell concentrate into ~20 microcentrifuge tubes.
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##Keep on ice until use (within the next two hours).
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#'''Add DNA:'''
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##Add 10μL of plasmid DNA to the 90μL of cell concentrate.
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##Keep on ice for 5 minutes.
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##Put 1mm cuvettes on ice too.
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#Electroporate:
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##Pipette the cell/DNA mixture into the cuvette
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##Electrporate at 1200 volts
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##Time constant should be ~5.0
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#Recovery:
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##Immediately transfer the electroporated cells to 900μL of MRS medium.
 +
##Incubate at 30°C for 2-3 hours.
 +
#Select:
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##Plate on medium with the appropriate antibiotic
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##Incubate at 30°C for two days.
 +
##Pick colony
==Notes==
==Notes==

Revision as of 14:59, 14 September 2011

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Contents

Overview

This is basically a modified version of the Mason (2005 protocol) for use with Lactobacillus plantarum and an Eppendorf 2510 electroporator.

Materials

  • MRS media
  • Glycine
  • Milipore water
  • 50mM EDTA solution
  • 0.5M Sucrose, 10% glycerol solution

°C==Procedure==

  1. Grow L. plantarum
    1. Inoculate 5ml MRS medium with L. plantarum freezer stock.
    2. Grow overnight at 30°C without shaking.
  2. Make 100ml 2% glycine:
    1. Add 1g glycine to two flasks of 50ml MRS medium.
    2. Shake to dissolve.
  3. Add 1ml overnight culture to each flask (1:50 dlution).
  4. Culture for ~3 hours at 37°C with shaking.
  5. Wash 2x with ice-cold DI water:
    1. Centrifuge culture for 5min at 4000g
    2. Resuspend in 25ml ice-cold DI water.
    3. Repeat.
  6. Treat with ice-cold EDTA:
    1. Centrifuge for 5min at 4000g
    2. Resuspend in 10ml 50mM EDTA.
    3. Incubate on ice for 5 minutes
  7. Wash with ice-cold DI water:
    1. Centrifuge culture for 5min at 4000g
    2. Resuspend in 25ml ice-cold DI water.
  8. Wash 2x with Electroporation Buffer:
    1. Centrifuge culture for 5min at 4000g
    2. Resuspend in 25ml ice-cold 0.5M Sucrose, 10% glycerol.
    3. Repeat.
  9. Concentrate cells:
    1. Centrifuge culture for 5min at 4000g
    2. Resuspend in 0.8ml ice-cold 0.5M Sucrose, 10% glycerol.
  10. Divide cells:
    1. Aliquot 90μL of cell concentrate into ~20 microcentrifuge tubes.
    2. Keep on ice until use (within the next two hours).
  11. Add DNA:
    1. Add 10μL of plasmid DNA to the 90μL of cell concentrate.
    2. Keep on ice for 5 minutes.
    3. Put 1mm cuvettes on ice too.
  12. Electroporate:
    1. Pipette the cell/DNA mixture into the cuvette
    2. Electrporate at 1200 volts
    3. Time constant should be ~5.0
  13. Recovery:
    1. Immediately transfer the electroporated cells to 900μL of MRS medium.
    2. Incubate at 30°C for 2-3 hours.
  14. Select:
    1. Plate on medium with the appropriate antibiotic
    2. Incubate at 30°C for two days.
    3. Pick colony

Notes

All questions, input and feedback are welcome!

References

There will be one here soon.


Contact

  • mas853@psu.edu

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